Transcriptional Coactivator Protein p300

Abstract

The p300/cAMP response element-binding proteinbinding protein (CBP) family members include human p300 and cAMP response element-binding protein-binding protein, which are both important transcriptional coactivators and histone acetyltransferases. Although the role of these enzymes in transcriptional regulation has been extensively documented, the molecular mechanisms of p300 and CBP histone acetyltransferase catalysis are poorly understood. Herein, we describe the first detailed kinetic characterization of p300 using fulllength purified recombinant enzyme. These studies have employed peptide substrates to systematically examine the substrate specificity requirements and the kinetic mechanism of this enzyme. The importance of nearby positively charged residues in lysine targeting was demonstrated. The strict structural requirement of the lysine side chain was shown. The catalytic mechanism of p300 was shown to follow a ping-pong kinetic pathway and viscosity experiments revealed that product release and/or a conformational change were likely rate-limiting in catalysis. Detailed analysis of the p300 selective inhibitor Lys-CoA showed that it exhibited slow, tight-binding kinetics.Since the initial revelation that yeast Gcn5p (Gcn ϭ global control non-repressed) is a histone acetyltransferase (HAT) 1 (1), and the subsequent determination that a number of other transcriptional co-activators possess HAT activity, there has been an explosion of interest into the role of protein acetylation in vivo (reviewed in Refs. 2-5). More than 20 HATs have now been identified and these have been classified into one of five families (GNAT, MYST (MOZ, YBF2/Sas3, Sas2, Tip60), TAF II 250, nuclear receptor coactivators, and p300/cAMP response element-binding protein-binding protein (CBP)), primarily based upon amino acid sequence homology (2).The p300/CBP family of HATs is represented by two proteins, p300 and CBP, that share considerable sequence homology over their entire length; human p300 and CBP proteins share 91% sequence identity (6). The coactivator p300 is a 2414-amino acid protein that was initially identified as a nuclear binding target of the E1A oncoprotein (7), whereas CBP is 2441 amino acids long and was originally identified as a transcriptional coactivator that bound to phosphorylated cAMP response element-binding protein in mice (8). Subsequently, Arany et al. (6) noted the significant sequence homology between these two proteins and suggested that these two large proteins (ϳ265 kDa) might be functionally equivalent, which has since been confirmed in many cases (9). The protein pair, p300/CBP, as it is now commonly referred to, is important for both cell cycle progression and cellular differentiation and possesses a number of distinct functional domains that have been shown to interact with components of the RNA polymerase II holoenzyme, transcription factors, and nuclear hormone receptors and their co-activators (2, 10 -19) (Fig. 1). Following the determination that p300/CBP-associated factor is a bona fide HAT (2...

Acetylation/Deacetylation Modulates the Stability of DNA Replication Licensing Factor Cdt1

“…4). These lysines are conserved in humans, mice, and frogs, and both sites conform to the consensus for KAT3B substrates (18). Mutation of these sites has no effect on the ability of Cdt1 to bind to geminin (data not shown).…”

“…4D, compare lanes 4 and 6) or KAT3B (compare lanes 11 and 12). It should be noted that both of these sites are neighbored by another basic residue three or four amino acids downstream of the acetylation site, which is typical of KAT3B substrates (18). KAT2B prefers a basic or aromatic residue three amino acids downstream of the acetylation site, which only Lys 49 possesses (19).…”

Kinetic and Mass Spectrometric Analysis of p300 Histone Acetyltransferase Domain Autoacetylation

“…Autoacetylation-Previous studies revealed that p300-mediated acetylation of peptide substrates proceeds with apparent K m values for acetyl-CoA in the range of 1-30 M (and depends on the enzyme form and peptide substrate concentration) and is effectively blocked by Lys-CoA with a K i of 20 nM (24,25). We thus investigated the K m of acetyl-CoA for the autoacetylation process.…”

“…Moreover, recombinant protein prepared using standard expression methods has been complicated to study because of its tendency to undergo autoacetylation in vivo (24,25). These two obstacles have recently been largely overcome by using the technique of expressed protein ligation (26,27).…”

Multisubstrate adduct inhibitors: Drug design and biological tools

“…In addition, p300/CBP HAT activity appears to be enhanced in some types of cancer and, thus, selective p300/CBP HAT inhibitors may have utility as therapeutic agents [162][163][164] . Cole et al reported several studies of the structure and substrate processing, before designing bisubstrate analog inhibitors (Scheme 36) [164][165][166][167][168] . These bisubstrates were analogs with Lys-CoA, and studies of the substructures of the CoA moiety of Lys-CoA revealed that the CoA, without modification, was crucial 169 .…”